目的 建立酶法定点偶联制备抗体药物(antibody drug conjugate,ADC)抗表皮生长因子受体-2(human epidermal growth factor receptor 2,HER2)单抗-澳瑞他汀(MMAE)的质控方法。方法 建立适宜的方法对抗体偶联药物的纯度(分子大小变异体、电荷变异体)、偶联修饰位点和偶联比(antibodydrugratio,DAR)、结合活性和生物学活性、以及偶联过程工艺相关杂质[游离小分子药物和谷氨酰胺转移酶(microbial transglutaminase,mTG)残留]进行控制。结果 体积排阻色谱(SEC)纯度主峰为(98.35±0.07)%,非还原十二烷基硫酸钠毛细管凝胶电泳法(CE-SDS)完整蛋白比例为(95.64±0.07)%,还原轻链比例为(31.15±0.19)%,重链比例为(67.87±0.06)%,全柱成像毛细管电泳(iCIEF)主峰比例为(51.05±0.7)%,偶联反应工艺偶联率95%以上,LC-MS测定的DAR为1.93(完整相对分子量),RP-HPLC测定的DAR为2.0,HC:Q298的连接子毒素(linker and drug,LD)修饰比例近100%,样品相对生物学活性(相对于参比品计算出的相对活性)平均值在(101±6.7)%,相对结合活性平均值为(95±3.8)%,RP-HPLC测定游离小分子含量均低于定量下限15 ng,ELISA法测定残留mTG含量均低于定量下限为0.165 ng·mL-1。结论 初步建立了酶法定点偶联制备HER2单抗-MMAE偶联药物质控方法,该质控方法具有保证产品安全、有效、质量可控的特点,为酶法定点偶联制备抗体偶联药物的质量研究提供了参考依据。
Abstract
OBJECTIVE To establish quality control methods of an anti-human epidermal growth factor receptor 2(HER2)-MMAE(LD),a kind of enzymatic site-specific prepared antibody-drug conjugate(ADC). METHODS An orthogonal method was established to determine the purity(molecular size variants, charge variants), conjugation modification site, and antibody drug ratio(DAR), binding activity and biological activity, and process-related impurities(free small molecule drugs and MTG residues) in the coupling process. RESULTS The SEC peak monomer purity was(98.35±0.07)%, the NRCE-SDS monomer purity was (95.64±0.07)%, the RCE-SDS purity light chain ratio was(31.15±0.19)%, heavy chain ratio was(67.87±0.06)%, the iCIEF purity main peak ratio was(51.05±0.7)%, coupling reaction rate was above 95%,DAR were 1.93 and 2.0 respectively determined by LC-MS and RP-HPLC,LND1002 modification(HC: Q298)rate was nearly 100%,the average relative biological activities was (101±6.7)%, the average relative binding activity was(95±3.8)%,the free drug content was lower than 15 ng, and the residual mTG was lower than 0.165 ng·mL-1. CONCLUSION The methods for quality control of the product are cleveloped to ensure thequality safety and effectiveness, and provide a reference for the quality control of site-specific enzymatic coupling antibody-drug conjugates.
关键词
抗体偶联药物 /
定点酶法偶联 /
抗HER2单抗-MMAE偶联药物 /
药物抗体偶联比 /
质量研究
{{custom_keyword}} /
Key words
antibody-drug conjugate /
site-specific enzymatic coupling /
anti-human epidermal growth factor receptor 2(HER2)-MMAE(LD) /
drug antibody coupling ratio /
quality control
{{custom_keyword}} /
中图分类号：
R917
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] SONG H B, LIU D L, LI P F, et al. Advanced research and technology development of antibody-drug conjugates[J]. Acta Pharm Sin(药学学报), 2019, 54(10): 1810-1817.
[2] XU S. Internalization, trafficking, intracellular processing and actions of antibody-drug conjugates[J]. Pharm Res(药物研究), 2015, 32(11): 3577-3583.
[3] ANNA W W, ADEUSZ R. Safety and tolerability of antibodydrug conjugates in cancer[J]. Drug Saf, 2019, 42(2): 295-314.
[4] HUI X W, CAO W R, ZHANG D, et al. Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase[J]. Chin J Biotechnol(生物工程学报), 2020, 36(4): 750-762.
[5] MARTIN G, RITA C, CARLO M B. Transglutaminases: nature′s biological glues[J]. Biochem J, 2002, 368(Pt 2): 377-396.
[6] YOKOYAMA K, NIO N, KIKUCHI Y. Properties and applications of microbial transglutaminase[J]. Appl Microbiol Biotechnol, 2004, 64(4): 447-454.
[7] ZHU Y, RINZEMA A, TRAMPER J, et al. Microbial transglutaminase-a review of its production and application in food processing[J]. Appl Microbiol Biotechnol, 1995, 44(3,4): 277-282.
[8] BAO Y L, PAN L. Research progress of mechanism and activity assay of microbial transglutaminase[J]. Sci Technol Food Ind(食品工业科技), 2008, 29(7): 265-269
[9] YOKOYAMA K, NIO N,KIKUCHI Y. Properties and applications of microbial transglutaminase[J].Appl Microbiol Biotechnol, 2004, 64(4): 447-454.
[10] DEWEID L, AVRUTINA O, KOLMAR H. Microbial transglutaminase for biotechnological and biomedical engineering[J]. Biol Chem, 2018, 400(3): 257-274.
[11] ZHOU J Q, HE T, WANG J W. Pegylation of cytochrome c at the level of lysine residues mediated by a microbial transglutaminase[J]. Biotechnol Lett, 2016, 38(7): 1121-1129.
[12] KHAMENEH B, JAAFARI M R, HASSANZADEH K M, et al. Preparation characterization and molecular modeling of pegylated human growth hormone with agonist activity[J]. Int J Biol Macromol, 2015, 80: 400-409.
[13] DENNLER P, CHIOTELLIS A, FISCHER E, et al. Transglutaminase-based chemo-enzymatic conjugation aprroach yields homogeneous antibody-drug conjugates[J]. Bioconjug Chem, 2014, 25(3): 569-578.
[14] ANAMI Y, XIONG W, GUI X, et al. Enzymatic conjugation using branched linkers for constructing homogeneous antibody-drug conjugates with high potency[J]. Org Biomol Chem, 2017, 15(26): 5635-5642.
[15] LIU B N, LUO J H. Research and development of innovative antibody-based drug[J]. Acta Pharm Sin(药学学报), 2017, 52(12):1811-1819.
[16] WANG L, XIA M, GAO K. Research progress and quality control of antibody-conjugated drugs[J].China Biotechnol(中国生物工程杂志), 2014, 34(4):85-94.
[17] WANG L, GUO SH, YU C F. Expert consensus on quality control and pre-clinical evaluation of antibody-drug conjugate[J]. Chin Pharm Aff(中国药事), 2018,32(7):993-1004.
[18] YI D, ALISON W, ROBIN E, et al. Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies[J]. Mabs, 2012, 4(5): 578-585.
[19] MARC B, CARL M, VERONICA J, et al. Predicting antibody developability profiles through early stage discovery screening[J]. Mabs, 2020, 12(1): 1-28.
[20] LI M, YU C F, WANG W B, et al. Development of methods for quality control of an anti-human epidermal growth factor receptor-2-MCC-DM1[J]. Chin J Pharm Anal(药物分析杂志), 2016,36(1):22-30.
[21] LI M, SUN L, ZHU L, et al. Quality control of anti-delta-like protein 3 antibody-MPVP-PBD[J]. Chin Pharm J(中国药学杂志), 2019, 54(24): 2018-2027.
[22] ADITYA W, YAN C, YATIN G, et al. Analytical methods for physicochemical characterization of antibody drug conjugates[J]. Mabs, 2011, 3(2): 161-172.
[23] CHIANG Z C, CHIU Y K, LEE C C, et al. Preparation and characterization of antibodydrug conjugates acting on HER2-positivecancer cells[J].PLoS One, 2020, 15(9): e0239813.
[24] BEHRENS C R, HA E H, CHINN L L, et al. Antibody-drug conjugates(ADCs) derived from interchain cysteine cross-linking demonstrate improved homogeneity and other pharmacological properties over conventional heterogeneous ADCs[J]. Mol Pharm, 2015, 12(11): 1-29.
[25] JUNUTULA JR, RAAB H, CLARK S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index[J]. Nat Biotechnol, 2008, 26(8): 925-932.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
“重大新药创制”国家科技重大专项课题资助(2019ZX09732002-005);河北省科技厅资助项目资助(20372402D)
{{custom_fund}}
PDF(1409 KB)
